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Observations concerning the persistence of living cells in maitland’s medium for the cultivation of vaccine virus

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Observations concerning the persistence of living cells in maitland’s medium for the cultivation of vaccine virus

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Abstract

Cells survive for at least 5 days and at times are capable of multiplying in a mixture of serum and Tyrode's solution used by Maitland for the cultivation in vitro of vaccine virus.

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... In 1929, it was demonstrated that the generation of virus material for eventual vaccine development and production was dependent upon the presence of viable host cells when tissue explants from hen kidneys were used as viral propagation tools [52]. Several decades of research and development were then necessary before the eventual replacement of animal tissue substrates by human diploid cell cultures, which became one of several standards in biotechnological manufacture for specific and quality-driven reasons [1,2,18,53]. ...
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Article
1. The virus of vaccinia in so called roller tube cultures of mixed embryonic chick tissue rapidly increases to maximal titre. 2. Under these conditions the quantity of virus in the tissue remains at or near the maximum for at least 9 weeks and considerable amounts are present in the fluids removed each day. 3. The same results are obtained when only fragments of embryonic chick heart are employed. 4. Many, though not necessarily all, of the cells in infected cultures remain alive and retain the capacity to proliferate. The presence of these living cells is essential for the persistence of the virus. 5. No apparent differences in the rate or amount of growth of cells in infected as contrasted with non-infected cultures can be discerned in the gross. 6. It is suggested, but not proved, that virus is continually being produced rather than simply preserved throughout the period.
Article
An analysis of some of the physiological factors active in Maitland tissue cultures has been presented in the hope that it may be of some value in clarifying the principles underlying tissue cultures in general. It has been found that the empirically determined necessity of using relatively small amounts of tissue in such cultures is dependent upon the fact that excessive tissue leads to a rapid change of reaction toward the acid side. Whereas tissue may remain viable in an environment as alkaline as pH 9 and over, viability is rapidly destroyed when the reaction approaches pH 6. Evidence is presented to indicate that the changes in electrode potentials which take place in Maitland cultures are not, as has been suggested, the determining factors upon which virus multiplication depends, although they may, of course, be incidentally important. It has been shown that there are fundamental differences between those conditions in Maitland cultures which favor the multiplication of a typical virus and those upon which the growth of the Rickettsiae of typhus fever depends. The virus which we have studied (equine encephalitis virus, western type) multiplies during the period of active tissue metabolism. The maximum virus titrations are obtained at about the time at which metabolism has come to a standstill. Thereafter the virus not only ceases to increase but rapidly deteriorates. The period of viability of the tissue cells themselves is shortened by several days in the presence of virus multiplication. There is some evidence that a temporary acceleration of oxygen uptake takes place during the time of active virus multiplication. Technical difficulties in controlling such experiments prevent certainty in regard to this point. In contrast with the conditions determining the growth of a virus agent in the Maitland cultures the multiplication of Rickettsiae does not begin to any determinable extent until after active cell metabolism has either become stabilized or has ceased. The Rickettsiae continue to grow at a time when the cells are no longer viable. It appears likely that these organisms find the most favorable conditions for growth in cells which are no longer metabolically active but in which some delicately heat-susceptible elements have not yet been disturbed. As a consequence of these observations, frozen and preserved embryonic tissues have been successfully used for Rickettsia cultivation. A report on these experiments will be made in a separate communication.
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A dermal strain of vaccine virus has been passed through 99 successive culture passages. This procedure led to a diminution in the pathogenicity of the active agent for the rabbit. By repeated testicular passages in rabbits, however, the virus regained its pathogenicity for that host. New cultures were initiated with the revived virus. A culture strain of virus that has been twice revived in this manner has remained fairly stable for the rabbit through 60 culture passages and it produces mild, yet effective vaccinal reactions in man. Virus in early cultures was not attenuated for man, but later cultures of the original strain and cultures of the 2nd and 3rd revived strains produced mild reactions without fever and discomfort to the patients. Intradermal vaccinations with the culture virus are safe and satisfactory. With the culture virus 118 infants and children have been inoculated and in 100 of them a positive reaction occurred. The culture virus produced a refractory state to a standard dermal strain of calf lymph and vice versa. Culture virus stored in 50 per cent neutral glycerol at -10 degrees C. or at +3 degrees C. maintained a considerable amount of its activity for at least 1 year. Desiccated culture virus sealed in tubes maintained some of its activity when stored at 37 degrees C. for 5 weeks. Fresh cultures can be initiated without difficulty from desiccated virus or from virus that has been stored with or without glycerol.
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1. Rickettsia prowazeki can be cultivated for many generations in vitro, without diminution in numbers or virulence, in media similar to those described by Maitland, Rivers, and others for the cultivation of certain viruses. In all probability, such cultures can be maintained indefinitely. 2. It has been impossible, thus far, to cultivate the typhus rickettsia without employing living tissue.
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1. A dermal strain of vaccine virus has been adapted to a simple culture medium consisting of minced chick embryo suspended in Tyrode's solution. 2. The bacteria-free culture virus, thus obtained, produces in lower animals and in man typical vaccinia that renders them refractory to infection with ordinary vaccine virus harvested from calves.
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The experiments in this paper show that testicle extract causes India ink particles and those of Prussian blue to spread much more extensively through the intercellular spaces than similar suspensions made with Ringer's solution. Methylene blue inoculated intravenously localizes more extensively in areas previously injected with testicle extracts than in control areas receiving injections of tissue extracts without enhancing power. Kidney extracts have this property to a less degree, whereas spleen extracts and blood serum are devoid of it. The spreading power of extracts is destroyed by heating at 60 degrees C. for 30 minutes, as is also the power to enhance infections. The precise mode of action of the Reynals factor is not known, but the results of the experiments here presented suggest that it may depend at least in part on the property whereby testicle extract increases the spread of injected material and alters the permeability of tissue cells. It is not inconceivable that changes in permeability facilitate the passage of vaccine virus through the endothelial cells of the blood and lymph vessels, and lead to the generalized vaccinia which is of frequent occurrence in the reported results (20). It has been shown that fluids and suspensions of inert particles are spread by the extract.B. tetanus and B. coli exotoxins and trypsin were not enhanced at all in their action despite the fact that they were spread through a more extensive area in the tissues. Viruses, on the other hand, are markedly influenced and in this respect resemble bacteria, not toxins and enzymes. It appears probable that a definite capacity for multiplication on the part of an injected substance is required if its pathogenic effects are to be enhanced. It may be concluded tentatively that the enhancing power of the testicle extract may depend on that property which not only spreads the injected material through a larger area but renders the tissue cells more easily penetrable by the agents.
Article
Vaccine virus, suspended in a mixture of serum and Tyrode's solution and separated by collodion membranes from a suspension of living kidney cells in serum and Tyrode's solution, remained active at 37 degrees C. for a longer period of time than did vaccine virus incubated only in a mixture of serum and Tyrode solution.
Article
THE cultivation of vaccinia virus in conjunction with growing tissue has been studied to a considerable extent. Parker (1923-24) and Parker and Nye (1925) were successful in propagating the virus through eleven generations and obtained an increase of 51,000 times. Small pieces of vaccine-infected rabbit's testis with rabbit's plasmiia were used and active proliferation of tissue took place regularly. Craciun and Oppenheimer (1926) separated the granular bodies described by Paschen (1906) from glycerinated calf-lymph. They claim to have cultivated these in embryonic guinea-pig cornea with guinea-pig plasma and Lock's solution on inverted coverslips in hollow-ground glass slides. These bodies survived for seventy-one days providing the cultures showed actively growing cells, but failed to survive if cultivated with dying or dead cells. This work has not yet been confirmed. Carrel and Rivers (1927) described experiments in which they obtained increase of the virus using finely minced chick enmbryo tissue. A strain of testicular virus was fixed to the tissue by standing for a period at cold roorn temperature. This virus-pulp emulsion was then mixed with Tyrode's solution and hen-plasma in suitable proportions and poured into Carrel " D " type culture flasks where clotting took place. After periods of incubation, varying from eight days to one month, the contents of these flasks were ground in a mortar and the supernatant fluid, suitably diluted, injected intradermally in rabbits. These tests showed an increase of 400 times after eight days' incubation. The technique was simple, and, in the opinion of the authors, could be adapted readily to the industrial production of vaccinia virus. Haagen (1928) cultivated the virus, using rabbit spleen and plasma in which was incorporated a very small piece of rabbit testicle. These were placed on mica coverslips over hollow-ground slides and incubated for three days, after which they were immersed for two or three minutes in a virus-containing fluid. The testicular implants were then placed in fresh plasma-spleen mixture and re-incubated. Thirty-seven subcultures were made and a thousandfold increase in virus obtained. In view of these investigations it was decided to study the problem more fully and to learn, if possible, something of the factors involved in the cultivation of viruses generally. The advantages of an apparently simple laboratory method of obtaining large quantities of vaccinia virus free from bacteria and tissue debris are obvious.
Article
A method has been developed by which the susceptibility of chickens to Rous virus can be tested, and the virulence of eight or ten different fluids compared in a single animal. The results of five series of experiments made with this technique can be summarized as follows: When a medium composed chiefly of chicken serum and Tyrode solution and containing no fresh tissues is inoculated with filtered extract of Rous and other sarcomas and incubated for 48 hours, it never produces a tumor after being injected into chickens. The virus has apparently been destroyed or at least has lost activity. But, in a solid medium, composed chiefly of serum and Tyrode solution and containing fragments of fresh tissues, the virus is found to increase readily, as shown in the first series of experiments. In the course of 15 months, the experiments have been repeated many times, with identical results. Flasks containing embryo pulp or leucocytes inoculated with filtered extract of sarcoma are used to keep on hand a constant supply of the Rous virus. The cultures of monocytes inoculated with the filtered extract often assume the appearance of the cultures of Rous sarcoma. They may also remain normal to all appearance despite the circumstance that the virus is multiplying within the medium. It is not certain that the activity of Rous virus is always accompanied by cell lesions, but there is no doubt that its increase depends on the presence of fresh tissues within the medium. The disappearance of the Rous virus from a medium that does not contain any fresh tissue may be interpreted as follows: the agent has been destroyed; or it is still present in a concentration lower than 1 in 50,000, which is the concentration required to produce a tumor even in the more susceptible chickens; or, according to the hypothesis of Gye, it is present in an inactive form. In the above experiments, the fresh tissues added to the medium might conceivably have enabled the virus to keep its full activity, through supplying the conditions requisite therefor, or they might merely have furnished an activating substance. The value of Gye's hypothesis was tested in a series of experiments. The results indicate that the tumor-producing virus present in the cultures was not composed of two parts, an inactive part multiplying in the medium, and an activating part supplied by the tissues. In another series of experiments, the relations between the reproduction of the virus and the quantity of the tissues contained in the medium were studied. The presence of a small fragment of leucocytic film or spleen tissue was sufficient to prevent the virus from disappearing. Approximately 1 c.mm. of spleen tissue in 3,000 c.mm. of medium may on occasion maintain a concentration of Rous virus in this fluid sufficient to produce a tumor upon inoculation into chickens. But this rarely happens. Generally when the medium contained only one fragment of spleen or leucocytic film, or 1 drop of embryonic pulp, the virus disappeared rapidly. When the quantity of tissue was from five to nine times larger, an abundant production of virus was practically always found. It became obvious that the quantity of active virus present in a medium containing multiplying cells depends upon the amount of tissue in the medium. In the fourth series of experiments, the kind of cells needed for the multiplication of the virus was ascertained. Rous virus was found to disappear rapidly from the fluid of cultures of fibroblasts, while it multiplied readily in cultures of leucocytes, the total volume of both tissues being approximately the same. It should be remembered that strains of fibroblasts obtained from Rous and other sarcomas very rarely produce tumors upon inoculation into chickens, while the inoculation of cultures of macrophages from the same tumors practically always determines their appearance. The fifth series of experiments showed that the cell metabolism is an important factor in the reproduction of the virus. When the activity of tissues had been suppressed or very much decreased by freezing, no virus was produced, while it multiplied readily in the control. The lack of oxygen for a period of 24 or 48 hours stopped cell proliferation, and at the same time the production of Rous virus ceased. However, the fluid of some of these dead or inactive cultures, after 6 days incubation, was still able to give rise to a small tumor upon inoculation into a chicken. There is an evident relation between the proliferating activity of the tissues and the production of the virus. But the agent may persist for several days in association with dead tissues. It may be concluded that the reproduction in vitro of the active virus depends on the presence of fresh tissues in the culture and upon the quantity, the activity, and the nature of the cells contained in the medium.
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